This invention relates to new diagnostic methods and diagnostic kits for the analysis of DNA using template-directed primer extension and highly sensitive detection by fluorescence polarization. This invention further relates to methods of identifying single nucleotide polymorphisms (SNPs) as well as other DNA sequence variations. Also, this invention relates broadly to the fields of biotechnology, molecular genetics and biomedicine.
DNA analysis is becoming increasingly important in the diagnosis of hereditary diseases, detection of infectious agents, tissue typing for histocompatability, identification of individuals in forensic and paternity testing, and monitoring the genetic makeup of plants and animals in agricultural research (Alford, R. L., et al., Curr Opn. Biotechnol (1994) 5:29-33). In addition, DNA analysis is crucial in large-scale genetic studies to identify susceptibility alleles associated with common diseases involving multiple genetic and environmental factors (Risch, N., et al., Science (1996) 273:1516-1517). Recently, attention is focused on single nucleotide polymorphisms (SNPs), the most common DNA sequence variation found in mammalian genomes (Cooper, D. N., et al., Hum Genet (1985) 69:201-205). While most of the SNPs do not give rise to detectable phenotypes, a significant fraction of them are disease-causing mutations responsible for genetic diseases. As the DNA sequence of the human genome is completely elucidated, large-scale DNA analysis will play a crucial role in determining the relationship between genotype (DNA sequence) and phenotype (disease and health) (Cooper, D. N., et al., Hum Genet (1988) 78:299-312). Although some assays have considerable promise for high throughput, the recently developed DNA diagnostic methods, including the high-density chip arrays for allele-specific hybridization analysis (Pease, A. C., et al., Proc Natl Acad Sci USA (1994) 91:5022-5026; Yershov, G., et al., Proc Natl Acad Sci USA (1996) 93:4913-4918), Wang, D. G., et al., Science (1998) 280:1077-1081, the homogeneous 5xe2x80x2-nuclease allele-specific oligonucleotide cleavage assay (TaqMan ASO, Livak, K. J., et al., Nat Genet (1995) 9:341-342), Whitcombe, D., et al., Clin Chem (1998) 44:918-923 a homogeneous fluorescence assay for PCR amplifications: its application to real-time, single-tube genotyping, the homogeneous template-directed dye-terminator incorporation (TDI) assay (Chen, X., et al., Nucleic Acids Res (1997) 25:347-353; Chen, X., et al., Proc Natl Acad Sci USA (1997) 94:10756-10761) the homogeneous dye-labeled oligonucleotide ligation (DOL) assay (Chen, X. et al. Genome Research (1998) 8: 549-556.), and the homogeneous molecular beacon ASO assay (Tyagi, S. et al. Nature Biotechnology (1998) 16: 49-53), all require specialty reagents and expensive detection instrumentation.
All the DNA diagnostic methods listed above involve amplification of target sequences to increase the sensitivity and specificity of the assays through polymerase chain reaction (PCR) or other similar amplification technologies. For example, one of the best known amplification methods is the polymerase chain reaction (referred to as PCR) which is described in detail in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,800,159. Briefly, in PCR, two primer sequences are prepared which are complementary to regions on opposite complementary strands of the target sequence. An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a thermostable DNA polymerase (e.g., Taq polymerase). If the target sequence is present in a sample, the primers will bind to the target and the polymerase will cause the primers to be extended along the target sequence by adding on nucleotides. By raising and lowering the temperature of the reaction mixture, the extended primers reaction products will dissociate from the target to become new targets. The excess primers will bind to the target and to the reaction products and the process is repeated. Other technologies result in amplification of a target sequence by strand displacement. These techniques include an enzymatic xe2x80x9cnickingxe2x80x9d or preferential cleavage of one of two strands present in a double-stranded DNA recognition site and the separation or detection of amplified products that include the target site, which is presented in U.S. Pat. Nos. 5,270,184. and 5,455,166 and each of which is hereby incorporated by reference herein.
Still other techniques employ the use of a fluorescently labeled primer and detect fluorescence or fluorescence polarization after the primer is hybridized to the target region, which is presented in U.S. Pat. No. 5,593,867. In U.S. Pat. No. 5,641,633, double-stranded DNA binding protein is also used to further preserve the hybridization of the fluorescently labeled primer to the target site. These methods require the use of fluorescently labeled primers and their detection while hybridized to the target site.
Template-directed primer extension is a dideoxy chain terminating DNA sequencing protocol designed to ascertain the nature of the one base immediately 3xe2x80x2 to the sequencing primer that is annealed to the target DNA immediately upstream from the polymorphic site. In the presence of DNA polymerase and the appropriate dideoxyribonucleoside triphosphate (ddNTP), the primer is extended specifically by one base as dictated by the target DNA sequence at the polymorphic site. By determining which ddNTP is incorporated, the allele(s) present in the target DNA can be inferred. This genotyping method has been widely used in many different formats and proven to be highly sensitive and specific (Syvanen, A.-C et al, Genomics (1990) 8: 684-692; Syvanen, A.-C. and Landegren, U. Human Mutation (1994) 3: 172-179).
Fluorescence polarization (FP) is based on the observation that when a fluorescent molecule is excited by plane-polarized light, it emits polarized fluorescent light into a fixed plane if the molecules remain stationary between excitation and emission (FIG. 1). Because the molecule rotates and tumbles in space during the fluorescent decay time, however, FP is not observed fully by an external detector. The observed FP of a fluorescent species is described by the Perrin equation and is related to the ratio of the rotational relaxation time and the fluorescent lifetime. If the temperature and viscosity are held constant the rotational relaxation time is proportional to the molecular volume of the fluorescent species. If local rotational motion of the fluorophore is minimal the FP is directly proportional to the molecular weight. In other words, when a fluorescent molecule and its conjugate are large (with high molecular weight), it rotates and tumbles slowly in solution compared to the fluorescent lifetime and FP is preserved. If the molecule is small (with low molecular weight), it rotates and tumbles faster and FP is largely lost (depolarized). The FP phenomenon has been used to study protein-DNA and protein-protein interactions (Dunkak, K. S. et al., Anal. Biochem. (1996) 243: 234-244; Heyduk, T. et al. Methods Enzymol. (1996) 274: 492-503; Wu, P. et al., Anal. Biochem. (1997) 249: 29-36), DNA detection by strand displacement amplification (Walker, G. T. et al., Nucleic Acids Res. (1996) 24: 348-353), and in genotyping by hybridization (Gibson, N. J. et al., Clin. Chem. (1997) 43: 1336-1341). More than 50 fluorescence polarization immunoassays (FPIA) are currently commercially available, many of which are routinely used in clinical laboratories for the measurement of therapeutics, metabolites, and drugs of abuse in biological fluids (Checovich, W. J., et al., Nature (1995) 375:254-256).
FP is expressed as the ratio of fluorescence detected in the vertical and horizontal axes and is therefore independent of the total fluorescence intensity. This is a clear advantage over other fluorescence detection methods in that as long as the fluorescence is above detection limits of the instrument used, FP is a reliable measure. The FP difference between totally bound and totally unbound fluorescent species represents the total dynamic range possible for the system. As long as a statistically significant difference can be experimentally derived by the interaction of a fluorophore attached to a low molecular weight species and its complexation or incorporation into a higher molecular weight species, FP represents a suitable detection scheme for the chemistry occurring in solution. This is normally empirically derived as local motions in the fluorescently-tagged species make it difficult to theoretically predict a suitable probe.
The total polarization reflects the sum of FP from all species in solution emitting at that wavelength. For a system in which the fluorophore is attached to a low molecular weight nucleotide producing a low polarization and is then incorporated into the probe oligomer at the allelic site, the polarization observed is described by the equation:
P=Pmax[ddNTP]b+Pmin([ddNTP]ixe2x88x92[ddNTP]b)
where Pmax is the polarization for dye-labeled ddNTP incorporated onto the TDI probe, Pmin is the polarization of the unincorporated dye-labeled ddNTP, [ddNTP]i is the initial concentration of dye-labeled ddNTP, and [ddNTP]b is the concentration of incorporated dye-labeled ddNTP. The maximum change in signal occurs with 100% incorporation of the ddNTP. Therefore, an important aspect in experimental design is to ensure that the initial concentration of dye-labeled ddNTP used in the reaction is kept at a minimum.
While the separate use of fluorescence polarization and TDI technologies have been reported, the effective use of these technologies together as disclosed herein has not been realized for many reasons. The sensitivity of instrumentation to enable the accurate observation of fluorescence polarization has significantly increased over the past several years. Additionally, the present inventors have conducted experimentation and development to refine the technologies and overcome initially negative observations that may have been seen by others leading them away from the present invention. The present invention is clearly novel and non-obvious over the prior teachings in the art because of the present invention""s ability to synthesize at the target site a fluorescently-labeled oligonucleotide comprising a fluorophore linked to a nucleotide and then detect fluorescence polarization of the fluorescently-labeled oligonucleotide in a host of ways which the present disclosure makes clear.
All publications cited are incorporated in their entirety by reference herein.
The present inventors have determined that the modification in the detection strategy for the TDI assay that they have developed allows for the rapid analysis of DNA sequence variations, including SNPs and unique insertions/deletions, in a homogeneous assay using an unmodified oligonucleotide probe, which eliminates the need for specialty reagents or expensive instruments. This present approach combines the specificity of enzymatic discrimination between the two alleles of a DNA sequence variation in a template-directed primer extension reaction and the high degree of sensitivity of fluorescence polarization.
This present method, designated template-directed dye-terminator incorporation assay with FP detection (xe2x80x9cFP-TDIxe2x80x9d assay; as presented in FIG. 2), provides that the sequencing primer is an unmodified primer with its 3xe2x80x2-end immediately upstream from the polymorphic or mutation site. The allele-specific dye-labeled ddNTP is incorporated onto the TDI primer in the presence of DNA polymerase and target DNA, when incubated in the presence of ddNTPs labeled with any fluorophore. Many fluorophores are readily available to the skilled artisan and include, for example, FAM (5-carboxy-fluorescein), ROX (6-carboxy-X-rhodamine), TMR (N,Nxe2x80x2,Nxe2x80x2-tetramethyl-6-carboxyrhodamine), and BODIPY dyes. The genotype of the target DNA molecule can be determined simply by exciting the dye on the sequencing primer and observing for FP (see FIG. 2).
As will be further disclosed herein, the present inventors have demonstrated that FP is a simple, highly sensitive, and specific detection method that may be used in a homogeneous primer extension genotyping assay. Both single-stranded synthetic DNA oligomers and double-stranded DNA fragments, including those amplified, for example, by the polymerase chain reaction (PCR, Saiki, R. K., et al., Science (1988) 239:487-491) may be used as templates in this assay. In all cases, the FP-TDI assay proves to be highly sensitive and specific.